Sandbox Reserved 322

Introduction


Arginase is a 105 kD homotrimeric metallo-protein, as shown in figure 1, and catalysis the hydrolysis of arginine to ornithine and urea by means of a binuclear spin-coupled Mn2+ cluster in the active site. Many organisms contain the enzyme arginase, for example Homo sapiens and Plasmodium falciparum, a parasite that causes cerebral malaria. In humans there are two forms of arginases that have evolved with differing tissue distributions and sub-cellular locations in mammals.

The two types of arginase is found in mammalian, are arginase I (hAI) and arginases II (hAII). Arginase I is found predominantly in the liver, where it catalyzes the final cytosolic step of the urea cycle. Arginases II is a mitochondrial enzyme that does not appear to function in the urea cycle and is more widely disturbed in numerous tissues, for example kidney, brains, skeletal muscle, mammary gland and penile corpus cavernosum. Recent studies show that Plasmodium falciparum arginase (PFA) plays a role in systemic depletion of arginine levels, which in turn has been associated with human cerebral malaria pathogenesis. In addition the arginase fold is part of the ureohydrolase superfamily, which also includes agmatinase, histone de-acetylase and acetylpolyamine amidohydrolase.

Structure and Function


In general arginase is a homotrimeric enzyme, which is present in the fifth and final step of the urea cycle for mammals. In humans, hAI converts L-arginine into L-orinithine and urea as shown in figure 2. Human arginase II plays a role in L-arginine homeostasis, by regulating L-arginine concentrations from cellular biosynthetic reactions such as nitric oxide (NO) biosynthesis. Additionally Plasmodium falciparum arginase is comparable to human arginase, due to the fact that it is 27% identical with human aginase I and II.

Overall arginase is a homotrimeric metallo-enzyme with a binuclear manganese MN cluster in each monomer as shown in the PDB identifier 3mmr. The overall fold of the arginase monomer belongs to the α/β protein class with a globular structure. One site of the active-site cleft is partially defined by the central 8-stranded β-sheet, and the metal binding sites is located on the edge of the β-sheet. The metal ion that is more deeply situated in the active-site cleft is designated Mn2+A while the other metal ion is designated Mn2+B. In Plasmodium falciparum arginase Mn2+A is coordinated by His 193, Asp 216, Asp 220, Asp 323 and a solvent molecule, with a square pyramidal geometry. The solvent molecule bridges both metal ions and also donates a hydrogen bond to Asp 220. Mn2+B is coordinated by His 218, Asp 216, Asp, 323, Asp 325 and the bridging solvent molecule in a distorted octahedral fashion. All metal ligands except for Asp 220 make hydrogen-bond interactions with other protein residues, and these interactions contribute to the stability of the metal binding site.

There are three different types of bridging metal ligands that facilitate the observed spin coupling between Mn2+A and Mn2+B. For the first ligand, the carboxylate side chain of Asp 216 is a syn-syn bidentate bridging ligand, with Oδ1 coordinated to Mn2+A and Oδ2 coordinated to Mn2+B. For the second ligand, the carboxylate side chain of Asp 323 is a monodentate bridging ligand, with Oδ1 coordinated to both Mn2+A and Mn2+B with anti- and syn-coordination stereo-chemistry, respectively. And finally the third ligand, is the solvent molecule bridges both manganese ion symmetrically. Also the Mn2+ ions coordinate with water, orienting and stabilizing the molecule and allowing water to act as a nucleophile and attack L-arginine, hydrolyzing it into orinithine and urea. Overall the two manganese metal ion in arginase maintain the proper function of the enzyme.

Mechanism


In general arginase is known to convert L-arginine into urea and L-ornithine, via hydrolysis, the proposed mechanism is adopted from Kanyo and colleagues as shown in figure 3. In the first step of the hydrolytic mechanism, Asp 220 stabilizes the metal-bridging hydroxide ion with a hydrogen bond during a nucleophilic attack at the guanidinium carbon of arginine. The resulting tetrahedral intermediate fall apart once a proton is transferred to the amino group of ornithine, and the proton transfer is mediated by Asp 220. It is proposed that His 233 shuttles a proton from bulk solvent to the ε-amino group of ornithine, before the product dissociation, as well a water molecule displaces urea. In addition, the metal coordination facilitates the ionization of this water molecule to regenerate a nucleophilic hydroxide ion. During this process a proton transfer occurs to the bulk solvent and is mediated by shuttle-group His 233.

Arginase and the Physiology of Sexual Arousal
Female sexual arousal disorder is defined as an inability to achieve or maintain sufficient sexual excitement, including clitoral erection and genital engorgement, and it is a physiologically analogous to male erectile dysfunction, which is defined as a deficiency in genital blood circulation which compromises the hemodynamic of erectons. Nitric oxide (NO) is the principle mediator of erectile functions and governs nonadrenergic, noncholinergic neurotransmission in penile corpus cavernosum smooth muscle. NO cause’s rapid relaxation of smooth muscle tissue and thereby facilitates the engorgement of the corpus cavernosum. Thus, NO synthase is a critical enzyme in the physiology of sexual arousal. Also, human arginase II is a critical enzyme in the physiology of sexual arousal, due to the fact it coexpressed with NO synthase in smooth muscle tissue. Given that hAII and NO synthase compete for the same substrate L-arginine as shown in figure 4, arginase appears to attenuate NO synthase activity and NO-dependent smooth muscle relaxation by depleting the substrate pool of L-arginine that would be available to NO synthase. In addition arginase is inhibited by the boronic acid inhibitor (ABH ), which maintains L-arginine concentrations, which in turn enhances NO synthase activity and NO-dependent smooth muscle relaxation in tissue. Thus over expression of human arginase II contributes to erectile dysfunction, and human penile arginase is a potential target for the treatment of male sexual dysfunction.